Development of RP-HPLC method for simultaneous estimation of gemifloxacin and ambroxol in tablet formulation

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Pharmacreations | Vol.2 | Issue 2 | April-June-2015
Journal Home page: www.pharmacreations.com
Research article Open Access
Development of RP-HPLC method for simultaneous estimation of
gemifloxacin and ambroxol in tablet formulation
Mohamed H. Assaleh*, Fathi H. Assaleh
Department of Chemistry, Faculty of Science, University of Zawia, Az-Zawiyah, Libya.
* Corresponding author: Mohamed H. Assaleh
E-mail id: mohammed_assaleh@icloud.com
ABSTRACT
A reverse phase high performance liquid chromatographic method was developed for the simultaneous
determination of gemifloxacin and ambroxol in tablet dosage forms. Separation of both gemifloxacin and ambroxol
was achieved within 7 min with required resolution, accuracy and precision thus enabling the utility of the method
for routine analysis. Chromatographic separation was achieved on a BDS Hypersil C18 column (250 × 4.6 mm, 3.5μ)
using a mobile phase consisting of phosphate buffer pH 5.8 and acetonitrile in the ratio of 40:60 at a flow rate of 1.2
mL per min. The detection was made at 246 nm. The retention time of gemifloxacin and ambroxol were 2.553 and
3.546 min respectively. The method was found linear over the range of 50-90 μg/mL for gemifloxacin and 11.7-21
μg/mL for ambroxol. The proposed method was validated as per the ICH guidelines.
Keywords: Gemifloxacin, Ambroxol, HPLC, Validation.
INTRODUCTION
Gemifloxacin is a quinolone class of antibacterial
agent.
Chemically, it is known 7-[(4Z)-3-(amino
methyl)-4-(methoxyimino) pyrrolidin-1-yl]-1-cyclo
propyl-6-fluoro-4-oxo-1,4-dihydro-1,8 naphthyridine
-3-carboxylic acid. The bactericidal action of
gemifloxacin results from inhibition of the enzymes
topoisomerase II (DNA gyrase) and topoisomerase
IV, which are required for bacterial DNA replication,
transcription, repair, and recombination.1-3
Ambroxol is mucolytic agent. Chemically, it is
known as trans-4-(2-Amino-3,5-dibrombenzyl
amino)-cyclohexanol. Ambroxol is a metabolite of
bromhexine and is very potent inhibitor of the
neuronal Na+ channels, with secretolytic and
secretomotoric actions that restore the physiological
clearance mechanisms of the respiratory tract, which
play an important role in the body’s natural defence
mechanisms. It stimulates synthesis and release of
surfactant by type II pneumocytes. Surfactant acts as
an anti-glue factor by reducing the adhesion of mucus
to the bronchial wall, in improving its transport and
in providing protection against infection and irritating
agents4-6
The literature survey revealed that there are two
HPLC methods8,9
and the other methods like
HPTLC10
and UV12-14
Spectroscopic methods
available for the determination of gemifloxacin and
ambroxol in their combined dosage forms. Various
HPLC methods 15,16
were developed for estimation of
drugs in their individual dosage forms. Hence there is
a need to develop a method. The present study was
aimed to develop a new simple, precise, accurate,
robust method for simultaneous estimation of
gemifloxacin and ambroxol in combined
pharmaceutical dosage form.
Journal of Pharmacreations

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Fig. 1. Structures of gemifloxacin and ambroxol
EXPERIMENTAL
Instrumentation
The Waters HPLC system equipped with auto
sampler and UV or DAD was used for method
development, and method validation. The output
signal was monitored and processed by using
Empower software.
Materials
Gemifloxacin and ambroxol bulk drugs were made
available from Pharmatrain, Hyderabad,
orthophosphoric acid, methanol, acetonitrile were
obtained from Merk. Commercially available
gemifloxacin and ambroxol tablets were used for the
dosage form analysis. All chemicals and reagent used
were of HPLC grade, Milli-Q-water was used
throughout the experiment. Commercially available
gemifloxacin and ambroxol dosage form was used for
the study.
Chromatographic conditions
The HPLC system was operated isocratically at flow
rate of 1.2 mL/min. The mobile phase found to be
most suitable for analysis was phosphate buffer (pH
5.8): acetonitrile in the ratio of 40: 60% v/v, detection
was carried out at 246 nm. The separation was
carried out on BDS Hypersil C18 (4.6 x 250 mm, 3.5
m).
Preparation of standard stock solution
Accurately weigh and transfer 10 mg of
Gemifloxacin & 10mg of Ambroxol working
standard into two separate 100 mL clean dry
volumetric flask add Diluent and sonicate to dissolve
it completely and make volume up to the mark with
the same solvent (Stock solution). Further pipette 7.0
mL of Gemifloxacin & 1.64 mL Ambroxol of the
above stock solution into a 10 mL volumetric flask
and dilute up to the mark with diluent.
Assay of Pharmaceutical Dosage form:
(Sample Preparation)
Twenty tablets were weighed to get the average
weight and then ground. An amount of powder
equivalent to 10 mg was transferred to a 100 mL
volumetric flask, added 70 mL of methanol and
sonicated for 10 min with intermediate shaking,
followed by making up to volume with methanol to
obtain a solution containing 100 mg/mL of
gemifloxacin and 100 mg/mL of ambroxol. Further
pipetted a 7.0 mL of the above stock solution into a
10 mL volumetric flask and dilute up to the mark
with diluent.
RESULTS AND DISCUSSION
Method development
Chromatographic parameters were preliminary
optimized to develop a LC method for simultaneous
determination of gemifloxacin and ambroxol with
short analyses time (< 7 min), and acceptable
resolution (> 2). The isoabsortive point of
gemifloxacin and ambroxol selected was 246 nm. In
order to identify a suitable organic modifier, various
compositions of acetonitrile and methanol were
tested. Methanol produced a high retention time for
Simvastatin and high column pressures due to the
high viscosity. Acetonitrile was found to display
advantageous separations. Change of percentage of
acetonitrile in the mobile phase brought about a great
influence on retention time of the two drugs. Effect
of mobile phase pH on two drugs was investigated at
a pH of 3, 4.5, and 5.8. Different columns like X-
terra, Inertsil, Hypersil columns were tried.

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Finally separation for simultaneous determination of
gemifloxacin and ambroxol was carried out by
isocratic elution using 40% phosphate buffer (pH 5.8)
and 60% acetonitrile with a flow rate of 1.2 mL/min
using BDS Hypersil C18 (4.6 x 250 mm, 3.5 m).The
system suitability parameters are summarized in
Table-1 and a typical chromatogram is shown in Fig.
2.
Fig.2. Standard chromatogram of gemifloxacin and ambroxol
Method validation
The above method was validated according to ICH
guidelines to establish the performance
characteristics of a method (expressed in terms of
analytical parameters) to meet the requirements for
the intended application of the method7
.
Linearity
The linearity of an analytical procedure is its ability
(within a given range) to obtain test results which are
directly proportional to the concentration (amount) of
analyte in the sample. Linearity of detector response
for gemifloxacin and ambroxol was established by
analyzing serial dilutions of a stock solution of the
working standard. Five concentrations ranging from
50-90 µg/mL for Gemifloxacin and 11.7-21 µg/mL
for Ambroxol were prepared and analyzed. The
linearity graph was plotted using concentration Vs
peak area and they were shown in Figs. 3 and 4.
Slope, correlation coefficient (R) and intercept were
calculated and the results were shown in Table-1.
Precision and Intermediate precision
For the precision study, repeatability study was
carried out for short time interval under the same
chromatographic conditions. For the intermediate
precision study, repeatability study was carried out in
different day under the same chromatographic
conditions. The sample was injected in five replicate.
The peak area for all the five replicate was recorded.
The mean and % relative standard deviation (%RSD)
was calculated. From the data obtained the developed
RP-HPLC method was found to be precise.
Accuracy
The accuracy of the method was determined by
recovery experiments. Known concentration of
working standard was added to the fixed
concentration of the pre-analyzed tablet solution.
Percent recovery was calculated by comparing the
area before and after the addition of working
standard. For both the drugs, recovery was performed
in the same way. The recovery studies were
performed in triplicate. This standard addition
method was performed at 50%, 100%, 150% level
and the percentage recovery was calculated. The
results are shown in Table-1.
Robustness
Robustness of the method was checked by making
slight deliberate changes in chromatographic
conditions like mobile phase ratio, flow rate. It was
observed that there were no marked changes in
chromatograms, which demonstrated that the
developed RP-HPLC method is robust.
LOD and LOQ
The LOD and LOQ of gemifloxacin and ambroxol
were determined by using the signal to noise
approach as defined in ICH guidelines. The

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concentration with signal to noise ratio of at least 3
was taken as LOD and concentration with signal to
noise ratio of at least 10 was taken as LOQ. The
results are shown in Table-1.
Assay of pharmaceutical formulation
The proposed validated method was successfully
applied to determine gemifloxacin and ambroxol in
their tablet dosage form. The assay of gemifloxacin
and ambroxol was found to be 99.75 ±0.83 and
101.52 ±0.49 respectively. The result obtained for
gemifloxacin and ambroxol was comparable with the
corresponding labeled amounts and they were shown
in Table-1.
CONCLUSIONS
The proposed RP-HPLC method allows for accurate,
precise, and reliable measurement of gemifloxacin
and ambroxol simultaneously in combined dosage
form. The developed RP-HPLC method was found to
be simple, rapid, accurate and precise for the
concurrent estimation of drugs in respective two-
component tablet dosage form of gemifloxacin and
ambroxol. The RSD for all parameters was found to
be less than two, which indicates the validity of
method and assay results obtained by this method are
in fair agreement. The developed method can be used
for routine quantitative simultaneous estimation of
gemifloxacin and ambroxol in multicomponent
pharmaceutical preparation.
Table 1: Results for gemifloxacin and ambroxol
System Suitability Parameters Gemifloxacin Ambroxol
Retention Time (min) 2.553 3.546
USP Plate Count 2265 4067
USP Tailing Factor 1.71 1.41
USP Resolution 4.55
Validation Parameters
Assay (%) 101.22 101.92
Linearity Range (µg/mL) 50-90 11.7-21
Correlation coefficient 0.999 0.998
Precision (%RSD) 0.19 0.2
ID precision (%RSD) 0.14 0.2
Accuracy (% Recovery) 100.08 100.59
LOD (µg/mL) 0.033 0.028
LOQ (µg/mL) 0.112 0.098
Fig. 3. Calibration curve for gemifloxacin
y = 20573x + 67582
R² = 0.999
0
500000
1000000
1500000
2000000
2500000
3000000
0 20 40 60 80 100
Area
Concentration (µg/ml)
Gemifloxacin

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Fig. 4. Calibration curve for ambroxol
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y = 25392x + 18694
R² = 0.998
0
200000
400000
600000
800000
0 5 10 15 20 25
Area
Concentration (µg/ml)
Ambroxol

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